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J Mol Recognit. 2009 Nov-Dec;22(6):446-52. doi: 10.1002/jmr.962.

Binding kinetics and activity of human poly(ADP-ribose) polymerase-1 on oligo-deoxyribonucleotide substrates.

Author information

1
Departments of Radiation Medicine, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, DC 20057, USA. tjorge01@georgetown.edu

Abstract

Poly(ADP-ribose) polymerase-1 (PARP-1) is a mammalian enzyme that attaches long branching chains of ADP-ribose to specific nuclear proteins, including itself. Because its activity in vitro is dependent upon interaction with broken DNA, it has been postulated that PARP-1 plays an important role in DNA strand-break repair in vivo. The exact mechanism of binding to DNA and the structural determinants of binding remain to be defined, but regions of transition from single-stranded to double-strandedness may be important recognition sites. Here we employ surface plasmon resonance (SPR) to investigate this hypothesis. Oligodeoxynucleotide (ODN) substrates that mimic DNA with different degrees of single-strandedness were used for measurements of both PARP-1/DNA binding kinetics and PARP-1's enzyme activities. We found that binding correlated with activity, but was unrelated to single-strandedness of the ODN. Instead, PARP-1 binding and activity were highest on ODNs that modeled a DNA double-strand break (DSB). These results provide support for PARP-1 recognizing and binding DSBs in a manner that is independent of single-stranded features, and demonstrate the usefulness of SPR for simultaneously investigating both PARP-1 binding and PARP-1 auto-poly(ADP-ribosyl)ation activities within the same in vitro system.

PMID:
19585541
PMCID:
PMC3493158
DOI:
10.1002/jmr.962
[Indexed for MEDLINE]
Free PMC Article

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